Mesenchymal stem cells (MSCs) have the capacity to develop into a variety of tissue types including bone, cartilage, tendon, muscle, fat and hematopoietic-supportive stroma. Techniques for the isolation of human and animal MSCs have been described as have techniques for directing their differentiation into, inter alia, the osteogenic lineage. These cells have also been shown to retain their developmental potential following extensive subcultivation in vitro (Bruder et al., 1997b), supporting their characterization as stem cells.
Although several monoclonal antibodies (McAb) that react with the surface of human mesenchymal stem cells (hMSCs) in culture have been described (Haynesworth et al., 1992a), the molecular identity of their respective antigens was not addressed. Recently, a new series of McAbs directed against the surface of human MSCs undergoing osteoblastic differentiation was reported (Bruder et al., 1997a). One of these McAbs, referred to as SB-10, reacts with the surface of human MSCs during fetal long bone and calvarial development, as well as the surface of marrow-derived MSCs in culture prior to differentiation. Immunoreactivity is lost during lineage progression when the osteoblast marker alkaline phosphatase (APase) is expressed. The SB-10 antigen has been observed not only in the outer periosteum of developing bones, but also in the developing brain, lung, and esophagus (Bruder et al., 1997a).
In one aspect the invention provides a method for accelerating the rate of differentiation of human mesenchymal stem cells, which method comprises contacting such human mesenchymal stem cells with an agent that inhibits cell-to-cell interaction of such human mesenchymal stem cells. Preferably the agent disrupts cell-to-cell binding. Preferred embodiments include monovalent ligands for human mesenchymal stem cell surface molecules and polynucleotides complementary to nucleic acid sequences coding for such cell surface molecules.
In another aspect the invention provides a method for inhibiting the rate of differentiation of human mesenchymal stem cells, which method comprises contacting such human mesenchymal stem cells with an agent that stimulates cell-to-cell interaction of such human mesenchymal stem cells. Preferably the agent enhances cell-to-cell binding. Preferred embodiments include polyvalent ligands for human mesenchymal stem cell surface molecules.
Also contemplated is the genetic manipulation of human mesenchymal stem cells to augment their production of agents that modulate cell-to-cell interaction, such as homophilic surface molecules, e.g. ALCAM.
In arriving at the present invention, the inventors have isolated, by immunopurification, the SB-10 antigen, and identified it as activated leukocyte-cell adhesion molecule (ALCAM). Thus, in a particularly preferred embodiment, the invention provides a method for accelerating the osteogenesis of an ALCAM-bearing mesenchymal stem cell which comprises contacting said mesenchymal stem cell with a ligand that binds with ALCAM on a single mesenchymal stem cell. The use of whole antibody is not contemplated, as cell-cell-binding does not provide the acceleration of osteogenesis observed in accordance with the invention. Preferred embodiments include those where the ligand is an Fab fragment of an antibody to ALCAM (e.g. an Fab fragment of the monoclonal antibody produced by the hybridoma cell line of ATCC Accession No. HB 11789), a single chain antibody to ALCAM and an ALCAM or fragment thereof. Preferably, the MSCs are human and, most preferably, autologous to the intended recipient. Any of the agents that are known to induce commitment and differentiation into the osteogenic lineage are suitable for use with respect to the present invention.
The cell line culture identified as SB-10 has been deposited (in accordance with the requirements of the Budapest Treaty for patent purposes) with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 (deposit date Jan. 10, 1995), and is assigned the ATCC Accession number HB-11789.
In another aspect, the invention provides a method for accelerating the in vivo formation of bone from mesenchymal stem cells that have been exposed to an osteoinductive agent by contacting said mesenchymal stem cells with a ligand that binds with ALCAM on a single mesenchymal stem cell. The preferred embodiments are the same as those described for the above aspect.
In another aspect, the invention provides a composition comprising an ALCAM-bearing mesenchymal stem cell and a ligand that binds with ALCAM on a single mesenchymal stem cell. The preferred embodiments are the same as those for the above aspects.
In another aspect, the invention provides a method for accelerating the in vivo formation of bone in an individual in need thereof which comprises administering to said individual, at the site where such bone is to be formed, a composition comprising ALCAM-bearing mesenchymal stem cells and ligand that binds with ALCAM on a single mesenchymal stem cell. The preferred embodiments are the same as those for the above aspects.
In another aspect, the invention provides a composition comprising an ALCAM bearing mesenchymal stem cell, an agent that is osteoinductive for said mesenchymal stem cell and a ligand that binds with ALCAM on a single mesenchymal stem cell. This will produce bone either in vivo or in vitro. The preferred embodiments are the same as those for the above aspects.
In another aspect, the invention provides a method for accelerating the in vivo formation of bone in an individual in need thereof which comprises administering to said individual, at the site where such bone is to be formed, a composition comprising ALCAM-bearing mesenchymal stem cells, an agent that is osteoinductive for said mesenchymal stem cells and ligands that bind with ALCAM on a single mesenchymal stem cell. The preferred embodiments are the same as those for the above embodiments.